1. Field of the Invention
The invention relates to compounds effective in modulating cellular responses stimulated by ceramide-mediated signal transduction, in particular in response to stimulus by the cytokine tumor necrosis factor xcex1 (TNF-xcex1). More specifically, it relates to compounds which inhibit the development of conditions associated with cell stimulus through the ceramide-mediated signal transduction pathway.
2. History of the Prior Art
The sphingomyelin pathway is a cellular signal transduction pathway that is believed to be involved in mediating cellular responses to several cytokines (including TNF-xcex1 and IL-Ixcex2) and growth factors (e.g., platelet derived growth factor and fibroblast growth factor) (see, e.g., Dressier, et al., Science, 259:1715-1718, 1992; and, Jacobs and Kester, Amer.J.Physiol., 265: 740-747, 1993). It is believed that interaction of such molecules with cell surface receptors triggers activation of a plasma membrane sphingomyelinase. Sphingomyelinase in turn catalyzes the hydrolysis of sphingomyelin to deramide and phosphocholine. Ceramide is believed to act as a second messenger through activation of a proline-directed, serine/threonine kinase (ceramide-activated protein kinase or xe2x80x9cCaPKxe2x80x9d). Ceramide also interacts with MAP kinase and protein kinase C zeta (see, e.g., Rivas, et al., Blood, 83:2191-2197, 1993) and with a serine/threonine protein phosphatase (see, Hannun, et al., TIBS, 20:73-77, 1995).
Recent investigation has provided evidence that the sphingomyelin pathway may mediate cellular senescence and apoptosis (programmed cell death) in response to TNF-xcex1 (see, e.g., Jayadev, et al., J.Biol.Chem., 270:2047-2052, 1994; and, Dbaibo, et al., J.Biol.Chem., 268:17762-17766, 1993) and radiation (Haimovitz-Friedman, et al., J.Exp.Med., 180:525-535, 1994). In this respect, ceramide has been presumed to mimic the effects of TNF-xcex1 on intracellular processes.
The invention is directed toward the development and use of compounds to inhibit cellular responses to ceramide metabolites of the sphingomyelin signal transduction pathway, such as inflammation, fibrosis, ultraviolet light induced cutaneous immune suppression, cell senescence and apoptosis.
In one aspect, the invention consists of novel compounds comprised of heterocyclic molecules with biologically active side chains (the xe2x80x9ccompounds of the inventionxe2x80x9d). Purine, pteridine, thiadiazolopyrimidine, quinalozine and isoquinolone based compounds are included in the invention. Particularly preferred among these compounds are those which have enhanced water solubility; e.g., morpholinoethyl esters of the compounds of the invention.
The compounds of the invention do not inhibit the activity of cAMP phosphodiesterase and therefore do not pose the risk of side-effects associated with other TNF-xcex1 inhibitors (e.g., pentoxifylline), such as sleeplessness and anxiety. Indeed, surprisingly, one of the more potent TNF-xcex1 activity inhibitors among the compounds of the invention (compound 37) had the least inhibitory effect on phosphodiesterase type IV, the predominant phosphodiesterase isoenzyme in monocytes and neutrophils. This is due to the fact that compounds such as 37 do not have a methylxanthine structure. Many common phosphodiesterase inhibitors (such as theophylline, theobromine, and caffeine) are methylxanthine compounds.
Moreover, all of the compounds of the invention inhibit apoptosis and retard cellular responses to TNF-xcex1 in vitro and in vivo with greater potency than pentoxifylline. Unexpectedly, the potency of at least the non-isoquinolone compounds appears to be dependent in part on the presence of ring nitrogens (other than the pyrimidine nitrogens), suggesting that binding to the target receptor responsible for inhibition of the activity of TNF-xcex1 observed is also regulated to some extent by the presence of such ring nitrogens. Further, the effects of all of the compounds appear to be totally unrelated to phosphodiesterase inhibition. This is particularly interesting given that increases in cAMP levels in cells can induce apoptosis in B cells (see, e.g., Lxc3x8mo, et al., J.Immunol., 154:1634-1643, 1995).
Recently, studies have shown (see, e.g. Verheij, et al., Nature, 380:75-79, 1996) that ceramide initiates apoptosis through the stress-activated protein kinase (SAPK/JNK) pathway. Thus, cells exposed to stresses such as ionizing radiation, hydrogen peroxide, UV-C radiation, heat shock, and TNF-xcex1, or to C-2 ceramide, acquired biochemical and morphological features typical of apoptosis. Indeed, these stresses have been shown to increase cellular levels of ceramide. Moreover, the role of one of the stress-activated protein kinases, c-jun (referred to as Jun kinase or JNK) in apoptosis was confirmed following stress and C-2 ceramide exposure. In this regard, compounds of the invention, particularly the isoquinolines, have shown activity as inhibitors of Jun kinase activation induced by anisomycin (a non-specific protein synthesis inhibitor) in MOLT-4 human lymphoblastoid cells or by LPS in RAW mouse macrophages. Therefore, a possible mechanism of activity of these compounds is the inhibition of one arm of the stress response pathway involving a Jun kinase in response to stress or to ceramide.
Another aspect of the invention consists of methods for the use of the novel compounds in inhibiting ceramide-activated cellular responses to stimuli, in particular stimuli for cell senescence and apoptosis. This aspect of the invention has potential therapeutic significance in the treatment of cell death associated conditions such as stroke, cardiac ischemia, nerve damage and Alzheimer""s disease.
Another aspect of the invention consists of methods which exploit the ability of the compounds of the invention to absorb UW radiation. This aspect of the invention has potential therapeutic significance in the treatment and prevention of radiation dermatoses, including those associated with therapeutic regimes for treatment of cancer.
The compounds of the invention are expected to be particularly useful in reducing the effects of aging in skin as well as the onset and progression of radiation dermatitis.
In another aspect, the invention features a compound having the formula: 
where
R1 is a terminally substituted normal alkyl having from 1 to 7 carbon atoms, a terminally substituted alkenyl having from 2 to 7 carbon atoms, a terminally substituted ether having from 2 to 6 carbon atoms, a terminally substituted secondary amine having from 2 to 6 carbon atoms, or substituted aryl having less than 8 carbons, where said terminal group is NH2, substituted amino, acyloxy, SO3H, PO4H2, NNO(OH), SO2NH2, PO(OH)NH2, SO2R or COOR, where R is H, an alkyl having from 1 to 4 carbon atoms, an alkenyl having from 1 to 4 carbon atoms, tetrazolyl, benzyl, or an alkylamino, where the alkyl group has from 1 to 4 carbon atoms and the amino is NH2 or a substituted amino where the substituents on the amino have 1 to 6 carbon atoms, one of which can be replaced by an oxygen atom or nitrogen atom;
Z is C, CH, or N;
R2 is an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, or aralkyl having less than 7 carbon atoms when Z is C, R2 is a halogen, NO, amino, or substituted amino when Z is CH, or R2 is H, an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, or aralkyl having less than 7 carbon atoms when Z is N;
A is CO when Z is N, or CR5 when Z is C or CH, where R5 is H, an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, aryl or aralkyl having less than 7 carbon atoms, OH, or an O-alkyl having from 1 to 5 carbon atoms and there is a double bond between Z and A when Z is C and a single bond between Z and A when Z is CH;
Y1 is N, NR6, or CR6, where R6 is H, NO, an amino, a substituted amino, an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, or aralkyl having less than 7 carbon atoms;
Y2 is N or CH; and
X is S when Y1 and Y2 are N, CR7 when Y1 is NR6 where R7 is H, OH, SH, Br, Cl, or I, or xe2x95x90C(R3)xe2x80x94C(R4)xe2x95x90 when Y1 is N, CH or CR6 and Y2 is N or CH, where each of R3 and R4, independently, is H, an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, aryl, alkylcarboxyl, or aralkyl having less than 7 carbon atoms, SH, OH or an O-alkyl having from 1 to 5 carbon atoms,
or a salt thereof.
In another aspect, the invention features a compound having the formula: 
where Q is a halogen or substituted amino, R2 is a halogen, NO, an amino, or a substituted amino, R3 is SH or OH, R4 is SH or OH, R, is H, an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, aryl or aralkyl having less than 7 carbon atoms, OH, or an O-alkyl having from 1 to 5 carbon atoms, and R6 is NO, an amino, or a substituted amino,
or a salt thereof.
In preferred embodiments, the compound has the formula: 
Preferably, R1 is a terminally substituted normal alkyl having from 1 to 7 carbon atoms, where said terminal group and is NH2, substituted amino, or COOR, where R is H, an alkyl having from 1 to 4 carbon atoms, or an alkylamino, where the alkyl group has from 1 to 4 carbon atoms and the amino is a substituted amino where the substituents on the amino have 1 to 6 carbon atoms, one of which can be replaced by an oxygen atom or nitrogen atom; R2, when present, is an alkyl having less than 7 carbon atoms (i.e., Me, or xe2x80x94(CH2)2CH3); each of R3 and R4, independently, is H, SH, OH, an alkyl, aryl, alkylcarboxyl, or aralkyl having less than 7 carbon atoms, or an O-alkyl having from 1 to 5 carbon atoms; and R6 is NO, an amino, or a substituted amino (i.e., NHR, or NR2, where R is H, an alkyl having from 1 to 4 carbon atoms, an alkenyl having from 1 to 4 carbon atoms, tetrazolyl, benzyl, an alkylamino, where the alkyl group has from 1 to 4 carbon atoms and the amino is NH2 or a substituted amino where the substituents on the amino have 1 to 6 carbon atoms, one of which can be replaced by an oxygen atom or nitrogen atom, aryl, or substituted aryl, where the substituted aryl has one, two, or three substituents including halogens or alkyls having 1 to 4 carbons). More preferably, R1 is a terminally substituted normal alkyl having from 1 to 4 carbon atoms where said terminal group is COOR where R is an N-morpholinoalkyl group (i.e., an N-morpholinoethyl group), and each of R3 and R4, independently, is an O-alkyl having from 1 to 5 carbon atoms.
Most preferably, R1 is xe2x80x94(CH2)3COOR where R is an N-morpholinoethyl group, and R3 and R4 each are H or methoxy groups, thus providing the compounds with enhanced water solubility.
In other preferred embodiments, the compound has the formula: 
where
R1 is a terminally substituted normal alkyl having from 1 to 7 carbon atoms, a terminally substituted alkenyl having from 2 to 7 carbon atoms, a terminally substituted ether having from 2 to 6 carbon atoms, a terminally substituted secondary amine having from 2 to 6 carbon atoms, or substituted aryl having less than 8 carbons, where said terminal group is NH2, substituted amino, acyloxy, SO3H, PO4H2, NNO(OH), SO2NH2, PO(OH)NH2, SO2R or COOR, where R is H, an alkyl having from 1 to 4 carbon atoms, an alkenyl having from 1 to 4 carbon atoms, tetrazolyl, benzyl, or an alkylamino, where the alkyl group has from 1 to 4 carbon atoms and the amino is NH2 or a substituted amino where the substituents on the amino have 1 to 6 carbon atoms, one of which can be replaced by an oxygen atom or nitrogen atom;
R2 is H, an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, or aralkyl having less than 7 carbon atoms;
Y2 is N or CH;
R6 is H, an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, or aralkyl having less than 7 carbon atoms; and
R7 is H, OH, SH, Br, Cl, or I,
or a salt thereof.
Further advantages and embodiments of the invention included therein will become apparent from the following disclosure.